WO2014089811A1 - 一种酵母细胞固定化介质的制备方法及其应用 - Google Patents

一种酵母细胞固定化介质的制备方法及其应用 Download PDF

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WO2014089811A1
WO2014089811A1 PCT/CN2012/086600 CN2012086600W WO2014089811A1 WO 2014089811 A1 WO2014089811 A1 WO 2014089811A1 CN 2012086600 W CN2012086600 W CN 2012086600W WO 2014089811 A1 WO2014089811 A1 WO 2014089811A1
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fiber material
medium
fermentation
preparation
solution
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PCT/CN2012/086600
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English (en)
French (fr)
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应汉杰
陈勇
刘庆国
吴菁岚
陈晓春
谢婧婧
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南京工业大学
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Priority to PCT/CN2012/086600 priority Critical patent/WO2014089811A1/zh
Priority to US14/651,071 priority patent/US10144925B2/en
Publication of WO2014089811A1 publication Critical patent/WO2014089811A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C12N11/12—Cellulose or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M13/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment
    • D06M13/10—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment with compounds containing oxygen
    • D06M13/12—Aldehydes; Ketones
    • D06M13/123—Polyaldehydes; Polyketones
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M13/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment
    • D06M13/322—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment with compounds containing nitrogen
    • D06M13/325—Amines
    • D06M13/332—Di- or polyamines
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M13/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment
    • D06M13/322—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment with compounds containing nitrogen
    • D06M13/402—Amides imides, sulfamic acids
    • D06M13/418—Cyclic amides, e.g. lactams; Amides of oxalic acid
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/19—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with synthetic macromolecular compounds
    • D06M15/37—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • D06M15/59—Polyamides; Polyimides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention belongs to the field of industrial biotechnology, and relates to a preparation method of a yeast cell immobilization medium and an application thereof, and particularly relates to a preparation method of a yeast cell immobilization medium and a method for producing ethanol by using the medium. Background technique
  • ethanol As a renewable and clean energy source, ethanol has become a research hotspot in the field of industrial biotechnology.
  • the traditional ethanol fermentation process uses free cell fermentation, and the yeast continuously flows away with the fermentation mash, resulting in insufficient concentration of yeast cells in the fermenter, which makes the alcohol fermentation slow and the fermentation time is longer.
  • immobilized yeast has gained more and more attention and application in fuel ethanol fermentation production.
  • the essence of immobilized cell technology is to physically or chemically locate free cells in a defined spatial region, to maintain catalytic activity, and to be used repeatedly to reduce cell loss.
  • immobilized yeast fermentation has an impact on the physiological characteristics and metabolic activity of yeast cells, such as cell morphology, intracellular osmotic pressure and cell membrane permeability.
  • immobilized yeast can also increase the ability of yeast cells to withstand fermentation inhibitors and fermentation environments. Since the yeast cells are fixed on the carrier and a large number of yeast cells are aggregated, a certain concentration advantage is formed, thereby ensuring that the yeast is not contaminated by the bacteria.
  • the embedding method is the most commonly used and most widely studied immobilization method, that is, a method of trapping microbial cells in a network space gap of a water-insoluble multimeric compound to immobilize them.
  • the embedding method is generally classified into a natural carrier embedding method and an organic synthetic polymer carrier embedding method.
  • Naturally embedded carriers include agar, carrageenan, carrageenan, calcium alginate, sodium alginate and polyvinyl alcohol.
  • the natural embedding carrier has the advantages of non-toxicity, convenient molding and high immobilization density, but its mechanical strength is low, mass transfer is weak, and anti-microbial decomposition ability is poor; and organic synthetic polymer carriers such as polyacrylamide, polyvinyl alcohol, Although polyacrylic acid has high mechanical strength and stable chemical properties, the polymer network is formed under severe conditions during the immobilization process, which has a great damage to cells. On the other hand, since the cells are inside the material, there is a poor mass transfer and a late stage. The problem of bacterial death and low catalytic efficiency.
  • the adsorption method utilizes the ability of microorganisms to adsorb to the surface of solid materials or other cell surfaces, and adsorbs them on the surface of the water-insoluble carrier to immobilize them.
  • the adsorption method is divided into physical adsorption and ion adsorption.
  • Physical adsorption is the use of substances with high adsorption capacity such as silica gel and activated carbon to immobilize microorganisms, while ion adsorption uses electrostatic action to adsorb microbial cells on ion exchangers.
  • the advantage of the adsorption method is that the method operates the cartridge, the properties are stable and difficult to decompose, and the effect on the cell activity is small, but the disadvantage is that the adsorbent material in the method has poor reusability.
  • the object of the present invention is to provide a novel yeast cell surface immobilization medium which utilizes a fiber material to immobilize yeast cells, thereby overcoming the deficiencies of the prior art.
  • the process is stable, non-toxic, the adsorption effect is good, and it has a good protection and promotion effect on cell activity and growth. Fermentation of ethanol by the yeast cells fixed by the medium can not only effectively ensure stable fermentation of ethanol, high-efficiency continuous production, but also reduce energy consumption in the ethanol separation process.
  • a method for preparing a yeast cell immobilization medium comprising the steps of:
  • the surfactant is selected from one or more of polyetherimide, diethylenetriamine, diammonium propylamine, polyethyleneimine, and succinimide. ;
  • the fiber material is selected from cotton fiber fabric; activated carbon fiber; polyester fiber; silk; bamboo fiber; polyvinyl alcohol fiber, such as vinylon; Spandex, acrylic; polyurethane foam; one or more of bagasse and corn stover.
  • the concentration of the hydrochloric acid is 0.5 to 2 mol/L.
  • the crosslinking agent is selected from the group consisting of glutaraldehyde, glyoxal, succinaldehyde, 2-mercapto-1,3-propanedialdehyde, and (2S,3R)-2,3- One or more of dihydroxysuccinic aldehydes.
  • the support skeleton is a steel mesh.
  • the above preparation method comprises the following steps:
  • the fiber material is boiled in boiling water for 0.1-10 hours and then dried at 20-100 ° C, 4
  • the surfactant is selected from one or more of polyetherimide, diethylenetriamine, diammonium propylamine, polyethyleneimine, and succinimide.
  • the fiber material is laid on the support frame so as to be attached to the support frame and then rolled into a cylindrical shape.
  • the present invention also provides a yeast cell immobilization medium prepared by the above preparation method.
  • the present invention provides a method for fermentative production of ethanol using the yeast cell immobilization medium, the method comprising the steps of:
  • the yeast cells are cultured in a seed growth medium to obtain a seed liquid in a logarithmic growth phase, and the seed liquid is circulated through the above-mentioned yeast cell immobilization medium in the bioreactor, so that the yeast cells are fixed in the medium;
  • the seed growth medium comprises glucose 10-100 g/L, peptone 10-100 g/L, yeast extract 10-100 g/L, anhydrous magnesium sulfate 0.2-10 g/L, ammonium sulfate 0.1-10 g/ L, phosphate 0.5-15 g / L, pH 4-6;
  • the solution is discharged and added to the fermentation medium for cyclic fermentation; wherein the fermentation medium contains glucose 100-400 g/L, peptone 0.5- 20 g/L, yeast extract 0.5-20 g/L, ammonium sulfate 0.1-10 g/L, phosphate 0.5-10 g/L, ferrous sulfate heptahydrate 0.05-1 g/L, heptahydrate heptahydrate 0.05- 1 g/L, pH 4-6.5; or the fermentation medium is a cassava hydrolysate or a plant straw hydrolyzate containing 0.1-10 g/L of urea and 0.1-10 g/L of magnesium sulfate.
  • the above method comprises the steps of: (1) first culturing the yeast cells in a seed growth medium to obtain a seed liquid in a logarithmic growth phase, and then seeding the seeds at 30-42 °C.
  • the liquid is circulated through the above-mentioned yeast cell immobilization medium in the bioreactor, so that the yeast cells are fixed to the medium; wherein the flow rate of the seed liquid is 0.5-50 L/
  • the seed growth medium comprises glucose 10-100 g/L, peptone 10-100 g/L, yeast extract 10-100 g/L, anhydrous magnesium sulfate 0.2-10 g/L, ammonium sulfate 0.1-10 g/L, phosphate 0.5-15 g / L, pH is 4-6;
  • the fiber material filling amount in the bioreactor is 5-150 g / L;
  • the fermentation medium contains glucose 100-400 g/L, peptone 0.5-20 g/L, yeast extract 0.5-20 g/L, ammonium sulfate 0.1-10 g/L, phosphate 0.5-10 g/L, ferrous sulfate heptahydrate 0.05-1 g/L , the sulphuric acid heptahydrate is 0.05-1 g/L, and the pH is 4-6.5; or the fermentation medium is hydrolyzed by cassava hydrolysate or plant straw containing 0.1-10 g/L of urea and 0.1-10 g/L of magnesium sulfate. Liquid; The circulating flow rate of the fermentation broth is 0.5-50 L/hour.
  • the present invention achieves the following beneficial effects:
  • the immobilized material prepared by the invention has low cost, no toxicity, high mechanical strength, chemical inertness, no toxic effect on cells, no degradation by cells, no influence on growth and metabolism of microorganisms, and dead cells fall off by themselves. Living cells are able to self-proliferate and maintain high catalytic efficiency.
  • the fiber material has a high porosity (>95%) and a specific surface area (>40 m 2 /m 3 ), so that not only the immobilized cells have a high density (cell density up to 70 g/L), but also Good mass transfer performance, high mass transfer efficiency, more than half of fermentation time, ethanol fermentation time with glucose as substrate is reduced from 20 hours to 6-8 hours, using starchy food crops such as cassava, corn and molasses as substrates.
  • the ethanol fermentation time (sucrose, liquefaction, fermentation integration) also decreased from 30-50 hours to 8-15 hours, and the ethanol fermentation time with corn stalk, wheat straw, corn cob and other hydrolysates as substrates was also 30-40. The hour drops to 10-15 hours.
  • the immobilization medium of the invention can also improve the tolerance of the strain to ethanol, and the product concentration is raised from 90-110 g/L to 120-150 g/L under the condition of constant conversion rate, and the effluent bacteria The bulk density is very low, which significantly reduces the cost of separation in the later stages.
  • Figure 1 is a schematic view showing the operation of attaching a fiber material to a support frame (wire mesh);
  • Figure 2 is a schematic view showing a method for producing ethanol by fermentation using the yeast cell immobilization medium of the present invention
  • 1 represents a steel mesh
  • 2 represents a fibrous material
  • 3 represents a feed tank
  • 4 represents a pump
  • 6 denotes a product collection tank.
  • Example 1 Preparation of Ethanol from Glucose by Using Activated Carbon Fiber Cotton Surface Immobilization Medium
  • the activated carbon fiber cotton was placed in boiling water for 1 hour, and then dried in an oven at 80 ° C; then the dried fiber material was immersed in 10 g / L of succinimide solution for 2 hours, and washed with distilled water; then pre-treated fiber material in 10 g / L of cross-linking agent 2-mercapto-1,3-propanedialdehyde aqueous solution , after 2 hours of reaction, wash with distilled water; finally put 60-80! Dry in the oven.
  • the treated fiber material 1 and the steel mesh 2 are cut into the same size, and then the fiber material is laid flat on the steel wire mesh, and is rolled into a cylindrical shape, and the fiber material layer and the layer need to be loosely uniform, and The reactor 3 having a height to diameter ratio of 4 was charged.
  • the cultured strain is added to the feed tank 3, and the seed liquid is rapidly flowed from the bottom of the reactor 5 through the pump 4.
  • the feed pump 4 is closed, and the right circulation pump 4 is turned on. , fixed at a flow rate of 2 L/h for 36 h. Until the concentration of the cells in the feed tank 3 is small, the decrease is not significant.
  • the waste liquid is then discharged from the bottom of the reactor 5.
  • the fermentation medium (glucose concentration is 250 g/L) is rapidly fed through the feed tank 3.
  • the feed pump 3 is also closed, and the fermentation is carried out, wherein the temperature is 35 ° C, and the pH of the fermentation liquid is 4.
  • the flow rate is 10 L/h.
  • the cotton fiber fabric was boiled in boiling water for 1 hour, and then dried in an oven at 80 ° C; then the dried fiber material was immersed in a 15 g / L polyethyleneimine solution for 4 h, and distilled water was used. Washing; then pre-treating the fiber material in a 20 g/L cross-linking agent aqueous solution of glyoxal, after 4 h of reaction, washing with distilled water; finally, drying in an oven at 60-80 ° C.
  • the fermentation of the cassava liquid was carried out by enzymatic hydrolysis (the glucose after saccharification of the cassava liquid was about 245 g/L), wherein the temperature was 34 ° C and the pH of the fermentation broth was 4.5.
  • the flow rate is 20 L/h.
  • the glucose was almost depleted and the ethanol concentration reached 110 g/L.
  • Ferment 15 batches the average fermentation time was 20h, the average ethanol yield gl 1 , the average yield was 47%.
  • Example 3 Preparation of Ethanol from Fiber by Using Polyester Surface Immobilized Medium
  • the polyester is placed in boiling water for 1 hour, and then dried in an oven at 80 ° C; then the dried polyester is immersed in a 5 g / L polyethyleneimine solution for 3 h, and washed with distilled water; The initially treated polyester was immersed in a 15 g/L cross-linking agent glutaraldehyde aqueous solution, and after 2 hours of reaction, it was washed with distilled water; finally, it was placed in an oven at 60-80 ° C for drying.
  • the straw hydrolyzate was fermented according to the apparatus and method of Example 1 (the glucose in the hydrolyzate was about 110 g/L), wherein the temperature was 34 ° C, the pH of the fermentation broth was 4.5, and the flow rate was 27 L/h. After 14 hours of the first fermentation time, glucose was almost depleted and the ethanol concentration reached 52.85 g/L. 10 batches were fermented, the average fermentation time was 15 h, the average ethanol yield was 3.6 gL" 1 ⁇ 1 , and the average conversion rate was 47.3%.

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Abstract

提供一种酵母细胞固定化介质的制备方法，包括以下步骤：（1）将纤维材料在沸水中煮沸后烘干；（2）将纤维材料置于浓度为1〜100g/L的表面改性水溶液中浸泡，并用盐酸调节溶液的pH至7.0，再将纤维材料置于去离子水中充分漂洗后烘干；（3）将纤维材料置于浓度为1〜100g/L的交联剂水溶液中浸泡，再将纤维材料置于去离子水中充分漂洗后烘干；（4）将纤维材料附着于支撑骨架。还提供通过该制备方法所制备的酵母细胞固定化介质以及采用该酵母细胞固定化介质生产乙醇的方法。

Description

一种酵母细胞固定化介质的制备方法及其应用 技术领域
本发明属于工业生物技术领域， 涉及一种酵母细胞固定化介质的制备 方法及其应用， 具体涉及一种酵母细胞固定化介质的制备方法以及应用该 介质生产乙醇的方法。 背景技术
乙醇作为一种可再生的洁净能源， 已成为工业生物技术领域的研究热 点。 传统的乙醇发酵工艺， 采用游离细胞发酵， 酵母随发酵醪不断流走， 造成发酵罐中酵母细胞浓度不够大， 使酒精发酵速度慢， 发酵时间较长。
近年来， 固定化酵母在燃料乙醇发酵生产上得到了越来越广泛的关注 和应用。 固定化细胞技术本质就是用物理或化学的手段将游离细胞定位于 限定的空间区域， 使其保持催化活性， 并可反复使用， 减少细胞流失。 随 着固定化技术的发展， 对固定化方法和载体材料的研究越来越广泛。 研究 发现， 固定化酵母发酵对酵母细胞生理特性以及代谢活力等方面会产生影 响， 如细胞形态、 胞内渗透压以及细胞膜通透性等。 同时， 固定化酵母还 可以提高酵母细胞耐受发酵抑制物与发酵环境的能力。 由于酵母细胞固定 后载体上聚有大量的酵母细胞， 形成了一定浓度优势， 从而保证酵母不受 杂菌的污染。
目前应用于酵母细胞的固定化方法越来越多， 目前常见的固定方法有 包埋法和吸附法。
包埋法是最为常用、 研究最广泛的固定化方法， 即一种将微生物细胞 截留在水不溶性多聚体化合物的网络空格空隙中使其固定化的方法。 包埋 法一般分为天然载体包埋法和有机合成高分子载体包埋法。 天然包埋载体 有琼脂、 角叉莱胶、 卡拉胶、 海藻酸钙、 海藻酸钠及聚乙烯醇等。 天然包 埋载体具有生物无毒、成型方便及固定化密度高等优点，但其机械强度低、 传质弱、 抗微生物分解能力较差； 而有机合成高分子载体如聚丙烯酰胺、 聚乙烯醇、 聚丙烯酸虽机械强度高、 化学性能稳定， 但在固定化过程中聚 合物网络形成条件剧烈， 对细胞有较大损害作用； 另一方面， 由于细胞在 材料内部， 所以存在着传质差、 后期菌体死亡、 催化效率低下的问题。 吸附法是利用微生物所具有的可吸附到固体物质表面或其他细胞表 面的能力， 将其吸附在水不溶性载体表面使其固定化。 吸附法分为物理吸 附和离子吸附。 物理吸附是使用硅胶、 活性炭等高吸附能力的物质来固定 微生物， 而离子吸附是利用静电作用使微生物细胞吸附在离子交换剂上。 吸附法优点在于这种方法操作筒单， 性质稳定不易分解， 对细胞活性影响 很小， 但缺点是该方法中的吸附材料反复使用性差。 发明内容
针对现有技术的不足之处， 本发明的目的在于提供一种新型的酵母细 胞表面固定化介质， 该介质利用纤维材料对酵母细胞进行固定化， 克服了 现有技术的不足， 该固定化方法不仅工艺稳定， 无毒性， 吸附效果好， 而 且对细胞活性和生长有很好的保护和促进作用。 采用该介质固定的酵母细 胞发酵生产乙醇， 不仅能有效保证乙醇稳定发酵、 高效连续生产， 而且可 以减小乙醇分离过程的能耗。
用于实现上述目的的技术方案如下：
一种酵母细胞固定化介质的制备方法， 该制备方法包括以下步骤：
( 1 )将纤维材料在沸水中煮沸后烘干；
( 2 )将纤维材料置于浓度为 l~100 g/L的表面改性剂水溶液中浸泡， 并用盐酸调节溶液的 pH至 7.0,再将纤维材料置于去离子水中充分漂洗后 烘干；
( 3 )将纤维材料置于浓度为 1~100 g/L的交联剂水溶液中浸泡， 再将 纤维材料置于去离子水中充分漂洗后烘干；
( 4 )将纤维材料附着于支撑骨架。
在上述制备方法中， 优选地， 所述表面活性剂选自聚醚酰亚胺、 二乙 三胺、 二曱胺基丙胺、 聚乙烯亚胺和丁二酰亚胺中的一种或多种；
在上述制备方法中， 优选地， 所述纤维材料选自棉纤维织物； 活性炭 纤维； 聚酯纤维； 丝绸； 竹纤维； 聚乙烯醇纤维， 例如维纶； 无纺布， 例 如涤纶、 丙纶、 锦纶、 氨纶、 腈纶； 聚氨酯泡沫； 甘蔗渣和玉米秸秆中的 一种或多种。
在上述制备方法中， 优选地， 所述盐酸的浓度为 0.5-2 mol/L。
在上述制备方法中， 优选地， 所述交联剂选自戊二醛、 乙二醛、 琥珀 醛、 2-曱基 -1,3-丙二醛和 （2S,3R ) -2,3-二羟基丁二醛中的一种或多种。 在上述制备方法中， 优选地， 所述支撑骨架为钢丝网。
就上述制备方法的一个具体实施方案而言， 上述制备方法包括以下步 骤：
( 1 )将纤维材料在沸水中煮沸 0.1-10 小时后在 20-100°C下烘干， 4
°C保存备用；
( 2 )将纤维材料置于浓度为 1~100 g/L 的表面改性剂水溶液中浸泡 1-50小时， 并用盐酸调节溶液的 pH至 7.0,再将纤维材料置于去离子水中 充分漂洗后 60°C烘干， 4°C保存备用；
( 3 )将纤维材料置于浓度为 1~100 g/L的交联剂水溶液中浸泡 1-50 小时， 再将纤维材料置于去离子水中充分漂洗后 60°C烘干， 4°C保存备用； 其中， 所述表面活性剂选自聚醚酰亚胺、 二乙三胺、 二曱胺基丙胺、 聚乙烯亚胺和丁二酰亚胺中的一种或多种。
( 4 ) 将纤维材料平铺在支撑骨架上， 使其附着于支撑骨架， 再卷成 筒状。
本发明还提供了上述制备方法制备的酵母细胞固定化介质。
另一方面， 本发明提供了一种采用所述酵母细胞固定化介质发酵生产 乙醇的方法， 该方法包括以下步骤：
( 1 ) 先将酵母细胞在种子生长培养基中培养至对数生长期获得种子 液， 再将种子液循环经过生物反应器中的上述酵母细胞固定化介质， 使得 酵母细胞固定于介质； 其中， 所述种子生长培养基包含葡萄糖 10-100 g/L, 蛋白胨 10-100 g/L ,酵母膏 10- 100 g/L ,无水硫酸镁 0.2- 10 g/L,硫酸铵 0.1-10 g/L, 磷酸盐 0.5-15 g/L, pH为 4-6;
( 2 ) 当生物反应器中的溶液的 OD值緩慢下降或小于 1 时， 排出溶 液， 加入发酵培养基进行循环发酵； 其中， 所述发酵培养基包含葡萄糖 100-400 g/L, 蛋白胨 0.5-20 g/L, 酵母膏 0.5-20 g/L, 硫酸铵 0.1-10 g/L, 磷酸盐 0.5-10 g/L, 七水合硫酸亚铁 0.05-1 g/L, 七水合硫酸辞 0.05-1 g/L, pH 4-6.5; 或者所述发酵培养基为包含尿素 0.1-10 g/L, 硫酸镁 0.1-10 g/L 的木薯水解液或植物秸秆水解液。
就上述方法的一个具体实施方案而言， 上述方法包括以下步骤： ( 1 ) 先将酵母细胞在种子生长培养基中培养至对数生长期获得种子 液， 再在 30-42 °C下将种子液循环经过生物反应器中的上述酵母细胞固定 化介质， 使得酵母细胞固定于介质； 其中， 所述种子液的流速为 0.5-50 L/ 小时， 所述种子生长培养基包含葡萄糖 10-100 g/L, 蛋白胨 10-100 g/L, 酵母膏 10- 100 g/L ,无水硫酸镁 0.2-10 g/L ,硫酸铵 0.1 - 10 g/L ,磷酸盐 0.5-15 g/L, pH为 4-6; 所述生物反应器中的纤维材料填充量为 5-150 g/L;
( 2 ) 当生物反应器中的溶液的 OD值緩慢下降或小于 1 时， 排出溶 液， 加入发酵培养基在 30-42 °C下进行循环发酵； 其中， 所述发酵培养基 包含葡萄糖 100-400 g/L, 蛋白胨 0.5-20 g/L, 酵母膏 0.5-20 g/L, 硫酸铵 0.1-10 g/L, 磷酸盐 0.5-10 g/L, 七水合硫酸亚铁 0.05-1 g/L, 七水合硫酸辞 0.05-1 g/L, pH为 4-6.5; 或者所述发酵培养基为包含尿素 0.1-10 g/L, 硫 酸镁 0.1-10 g/L 的木薯水解液或植物秸秆水解液； 发酵液的循环流速为 0.5-50 L/小时。
相对于现有技术， 本发明取得了如下的有益效果：
1、 本发明制备的固定化材料成本低， 无毒性， 机械强度高， 材料为 化学惰性， 对细胞不具有毒害作用， 也不会被细胞降解， 不影响微生物的 生长代谢， 死细胞自行脱落， 而活细胞能够实现自增殖过程， 始终维持很 高的催化效率。
2、 纤维材料具有较高的孔隙率 （>95% ) 和比表面积 （>40 m2/m3 ), 因此不仅固定化的细胞密度大（细胞密度最高可达 70 g/L ), 而且具有良 好的传质性能， 传质效率高， 发酵时间缩短一半以上， 以葡萄糖为底物的 乙醇发酵时间由 20小时下降至 6-8小时， 以木薯、 玉米、 糖蜜等淀粉类粮 食作物为底物的乙醇发酵时间 （糖化、 液化、 发酵一体化）也由 30-50小 时下降至 8-15小时， 以玉米秸秆、 小麦秸秆、 玉米芯等水解液作为底物的 乙醇发酵时间也由 30-40小时下降至 10-15小时。
3、 本发明的固定化介质还可以提高菌株对乙醇的耐受性， 在转化率 不变的条件下， 产物浓度由 90-110 g/L提升至 120-150 g/L, 并且流出液菌 体密度非常低， 大幅降低了后期的分离成本。 附图说明
以下， 结合附图来详细说明本发明的实施方案， 其中：
图 1为纤维材料附着于支撑骨架 （钢丝网） 的操作示意图；
图 2为采用本发明的酵母细胞固定化介质发酵生产乙醇的方法的示意 图；
其中， 1表示钢丝网； 2表示纤维材料； 3表示进料罐； 4表示泵； 5 表示生物反应器； 6表示产品收集罐。 实施发明的最佳方式
以下参照具体的实施例来说明本发明。 本领域技术人员能够理解， 这些 实施例仅用于说明本发明， 其不以任何方式限制本发明的范围。
下述实施例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施 例中所用的原料、 试剂等， 如无特殊说明， 均为市售购买产品。
实施例 1 采用活性碳纤维棉表面固定化介质由葡萄糖制备乙醇 首先， 将活性碳纤维棉放入沸水蒸煮 lh, 并随后置于 80°C的烘箱中 烘干；然后将烘干后的纤维材料浸泡在 10 g/L的丁二酰亚胺溶液中 2小时， 并用蒸馏水清洗；再将初步处理的纤维材料浸泡在 10 g/L的交联剂 2-曱基 -1,3-丙二醛水溶液中， 反应 2个小时后用蒸馏水清洗； 最后再置于 60-80 ！的烘箱里烘干。
参见图 1 , 将处理的纤维材料 1和钢丝网 2剪成大小相同的形状， 然 后将纤维材料平铺在钢丝网上， 与其卷成筒状， 纤维材料层与层之间需保 持疏松均匀， 并装入高与直径比为 4的反应器 3中。
然后将培养好的菌种加到进料罐 3中， 种子液通过泵 4从反应器 5底 部快速流进， 待种子液充满反应器 5后， 关闭进料泵 4, 打开右侧循环泵 4, 以流速为 2 L/h循环固定 36h。 直至进料罐 3中的菌体浓度很小， 降低 不明显。 然后从反应器 5底部排出废液。 再通过进料罐 3快速流加发酵培 养基（葡萄糖浓度为 250 g/L ), 充满反应器 5后， 同样关闭进料泵 3 , 进 行循环发酵， 其中温度为 35 °C , 发酵液 pH为 4, 流速为 10 L/h。 发酵时 间为 8h, 反应浓度达到 120 g/L, 比游离发酵缩短了 14h。 发酵 20批次， 平均发酵时间降至 7h, 乙醇产率达到 17.1 gl h-1 , 平均得率为 48%。 实施例 2 采用棉纤维织物表面固定化介质由木薯制备乙醇
首先， 将棉纤维织物放入沸水蒸煮 lh, 并随后置于 80°C的烘箱中烘 干； 然后将烘干后的纤维材料浸泡在 15 g/L的聚乙烯亚胺溶液中 4h, 并 用蒸馏水清洗；再将初步处理的纤维材料浸泡在 20 g/L的交联剂乙二醛水 溶液中， 反应 4h后用蒸馏水清洗； 最后再置于 60-80°C的烘箱里烘干。
发酵时按实施例 1中的装置及方法对木薯料液边酶解边发酵（木薯料 液糖化完后的葡萄糖为 245 g/L左右），其中温度为 34°C ,发酵液 pH为 4.5 , 流速为 20 L/h。第一批发酵 18h后，葡萄糖几乎耗尽，乙醇浓度达到 110 g/L。 发酵 15批次， 平均发酵时间为 20h, 乙醇平均产率 gl 1, 平均得率为 47%。 实施例 3 采用涤纶表面固定化介质制备由纤维制备乙醇
首先， 将涤纶放入沸水蒸煮 lh, 并随后置于 80°C的烘箱中烘干； 然 后将烘干后的涤纶浸泡在 5 g/L的聚乙烯亚胺溶液中 3h,并用蒸馏水清洗； 再将初步处理的涤纶浸泡在 15 g/L的交联剂戊二醛水溶液中， 反应 2h后 用蒸馏水清洗； 最后再置于 60-80°C的烘箱里烘干。
发酵时按实施例 1中的装置及方法发酵秸秆水解液（水解液中葡萄糖 为 110 g/L左右）， 其中温度为 34°C , 发酵液 pH为 4.5 , 流速为 27 L/h。 第一批发酵时间 14h后， 葡萄糖几乎耗尽， 乙醇浓度达到 52.85 g/L。 发酵 10批次， 平均发酵时间为 15h, 乙醇平均产率 3.6 gL"1^1 , 平均转化率为 47.3%。

Claims

权 利 要 求 书
1. 一种酵母细胞固定化介质的制备方法， 该制备方法包括以下步骤： ( 1 )将纤维材料在沸水中煮沸后烘干；
( 2 )将纤维材料置于浓度为 l~100 g/L的表面改性剂水溶液中浸泡， 并用盐酸调节溶液的 pH至 7.0,再将纤维材料置于去离子水中充分漂洗后 烘干；
( 3 )将纤维材料置于浓度为 1~100 g/L的交联剂水溶液中浸泡， 再将 纤维材料置于去离子水中充分漂洗后烘干；
( 4 )将纤维材料附着于支撑骨架。
2. 根据权利要求 1所述的制备方法， 其特征在于， 所述表面活性剂选 自聚醚酰亚胺、 二乙三胺、 二曱胺基丙胺、 聚乙烯亚胺和丁二酰亚胺中的 一种或多种。
3. 根据权利要求 1或 2所述的制备方法， 其特征在于， 所述纤维材料 选自棉纤维织物； 活性炭纤维； 聚酯纤维； 丝绸； 竹纤维； 聚乙烯醇纤维， 例如维纶； 无纺布， 例如涤纶、 丙纶、 锦纶、 氨纶、 腈纶； 聚氨酯泡沫； 甘蔗渣和玉米秸秆中的一种或多种。
4. 根据权利要求 1至 3中任一项所述的制备方法， 其特征在于， 所述 盐酸的浓度为 0.5-2 mol/L。
5. 根据权利要求 1至 4中任一项所述的制备方法， 其特征在于， 所述 交联剂选自戊二醛、 乙二醛、 琥珀醛、 2-曱基 -1,3-丙二醛和（2S,3R ) -2,3- 二羟基丁二醛中的一种或多种。
6. 根据权利要求 1至 5中任一项所述的制备方法， 其特征在于， 所述 支撑骨架为钢丝网。
7. 根据权利要求 1至 6中任一项所述的制备方法， 其特征在于， 所述 制备方法包括以下步骤：
( 1 )将纤维材料在沸水中煮沸 0.1-10 小时后在 20-100°C下烘干， 4 °c保存备用；
( 2 )将纤维材料置于浓度为 1~100 g/L 的表面改性剂水溶液中浸泡 1-50小时， 并用盐酸调节溶液的 pH至 7.0,再将纤维材料置于去离子水中 充分漂洗后 60°C烘干， 4°C保存备用；
( 3 )将纤维材料置于浓度为 1~100 g/L的交联剂水溶液中浸泡 1-50 小时， 再将纤维材料置于去离子水中充分漂洗后 60°C烘干， 4°C保存备用； 其中， 所述表面活性剂选自聚醚酰亚胺、 二乙三胺、 二曱胺基丙胺、 聚乙烯亚胺和丁二酰亚胺中的一种或多种；
( 4 ) 将纤维材料平铺在支撑骨架上， 使其附着于支撑骨架， 再卷成 筒状。
8. 根据权利要求 1至 7中任一项所述的制备方法制备的酵母细胞固定 化介质。
9. 一种采用酵母细胞固定化介质发酵生产乙醇的方法，该方法包括以 下步骤：
( 1 ) 先将酵母细胞在种子生长培养基中培养至对数生长期获得种子 固定化介质， 使得酵母细胞固定于介质； 其中， 所述种子生长培养基包含 葡萄糖 10-100 g/L, 蛋白胨 10-100 g/L, 酵母膏 10-100 g/L, 无水硫酸镁 0.2-10 g/L, 硫酸铵 0.1-10 g/L, 磷酸盐 0.5-15 g/L, pH为 4-6;
( 2 ) 当生物反应器中的溶液的 OD值緩慢下降或小于 1 时， 排出溶 液， 加入发酵培养基进行乙醇发酵； 其中， 所述发酵培养基包含葡萄糖 100-400 g/L, 蛋白胨 0.5-20 g/L, 酵母膏 0.5-20 g/L, 硫酸铵 0.1-10 g/L, 磷酸盐 0.5-10 g/L, 七水合硫酸亚铁 0.05-1 g/L, 七水合硫酸辞 0.05-1 g/L, pH为 4-6.5;或者所述发酵培养基为包含尿素 0.1-10 g/L,硫酸镁 0.1-10 g/L 的木薯水解液或植物秸秆水解液。
10. 根据权利要求 9所述的方法， 其特征在于， 该方法包括以下步骤： ( 1 ) 先将酵母细胞在种子生长培养基中培养至对数生长期获得种子 述的酵母细胞固定化介质， 使得酵母细胞固定于介质； 其中， 所述种子液 的流速为 0.5-50 L/小时， 所述种子生长培养基包含葡萄糖 10-100 g/L, 蛋 白胨 10-100 g/L,酵母膏 10-100 g/L,无水硫酸镁 0.2-10 g/L,硫酸铵 0.1-10 g/L, 磷酸盐 0.5-15 g/L, pH为 4-6; 所述生物反应器中的纤维材料填充量 为 5-150 g/L;
( 2 ) 当生物反应器中的溶液的 OD值緩慢下降或小于 1 时， 排出溶 液， 加入发酵培养基在 30-42°C下进行乙醇循环发酵； 其中， 所述发酵培 养基包含葡萄糖 100-400 g/L, 蛋白胨 0.5-20 g/L, 酵母膏 0.5-20 g/L, 硫酸 铵 0.1-10 g/L, 磷酸盐 0.5-10 g/L, 七水合硫酸亚铁 0.05-1 g/L, 七水合硫 酸辞 0.05-1 g/L, pH为 4-6.5; 或者所述发酵培养基为包含尿素 0.1-10 g/L, 硫酸镁 0.1-10 g/L的木薯水解液或植物秸秆水解液； 发酵液的循环流速为 0.5-50 L/小时。
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